Abstract: :
Purpose: Previous investigations have demonstrated the capabilityof the mammalian retina to synthesize melatonin. In addition,it has been shown that such synthesis is under the control ofa circadian oscillator located within the retina. Several studieshave shown that GABA modulates melatonin synthesis in the retina.An aim of the present investigation was to evaluate the possiblerole of GABA in the circadian mechanisms regulating melatoninsynthesisMethods: Rat retinas were cultured at constant temperature(33ºC) in darkness for 3 days using a flow-through apparatus.The culture medium (199) was supplemented with 100 microM ofGABA or GABA antagonists (saclofen and bicuculline). Perifusatedsamples were collected at 4 hour intervals, and melatonin levelswere measured by radioimmnoassay.Results: Control retinas releasedmelatonin with a clear circadian rhythm. Addition of 100 microMGABA to the medium did not affect the circadian rhythms of melatoninrelease; the same result was obtained culturing the retina with100 microM of saclofen (GABAB antagonist). In contrast, thecircadian rhythm of melatonin release/synthesis was abolishedwhen the retinas were cultured with 100 microM of bicuculline(GABAA antagonist).Conclusion: Our data show that 100 microMof the GABAA antagonist bicuculline inhibited melatonin releasein the rat retina, thus abolishing the circadian rhythm. Sucheffect could be a result of a direct action of GABA on the melatoninsynthesizing cells (i.e., the cone photoreceptors) or an indirectaction (i.e., throughout the increase in dopamine level whichin turn inhibits melatonin synthesis). As the first step tounderstand the mechanism by which GABA acts we are now usinglaser captured microdissection and single cell RT-PCR to investigateif GABA receptors are present within the melatonin synthesizingcells.